Vaccine immunotherapy for immune suppressed patients

ABSTRACT

A method for overcoming mild to moderate immune suppression includes the steps of inducing production of naïve T-cells and restoring T-cell immunity. A method of vaccine immunotherapy includes the steps of inducing production of naive T-cells and exposing the naive T-cells to endogenous or exogenous antigens at an appropriate site. Additionally, a method for unblocking immunization at a regional lymph node includes the steps of promoting differentiation and maturation of immature dendritic cells at a regional lymph node and allowing presentation of processed peptides by resulting mature dendritic cells, thus, for example, exposing tumor peptides to T-cells to gain immunization of the T-cells. Further, a method of treating cancer and other persistent lesions includes the steps of administering an effective amount of a natural cytokine mixture as an adjuvant to endogenous or exogenous administered antigen to the cancer or other persistent lesions.

CROSS-RELATED REFERENCE SECTION

[0001] This application claims the benefit of priority under 35 U.S.C.Section 119(e) of United States Provisional Patent Application No.60/243,912, filed Oct. 27, 2000, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The present invention relates to vaccine therapy for cancerpatients. More specifically, the present invention relates to a vaccineimmunotherapy which immunizes cancer patients, having immunesuppression, to both endogenous and exogenous tumor peptides orproteins.

[0004] 2. Background Art

[0005] It has become increasingly apparent that human cancers haveantigens which, if reacted upon by the host's immune systems, lead totumor regression. These antigens have been defined by both serologicaland cellular immune approaches. This has lead to the definition of bothB and T cell epitopes (Sahin U, et al, Curr Opin Immunol 9:709-715,1997; Van der Eynde, B, et al. Curr Opin Immunol 9:684-693, 1997; WangRF, et al., Immunologic Reviews 170:85-100, 1999). Based upon theseresults, it has become a goal of cancer immunotherapists to induceregressions of tumors. However, historically, successful efforts havebeen sporadic and generally minor in frequency and magnitude.

[0006] A fundamental problem in the effort to immunize cancer patientsis that the tumor-bearing state is associated with immunosuppressivemechanisms derived from both the tumor and the host's disturbed immunesystem (Kavanaugh D Y, et al, Hematol-Oncol Clinics of North Amer10(4):927-951, 1996), thereby making immunization difficult and untilnow impossible on a consistent basis. Immune suppression or depletioninvolves a reduced capacity of the immune system to respond. Suchsuppression can be drug or disease induced. The condition can be druginduced by treatment, virus induced as in AIDS, or induced by a diseasestate such as cancer. The immune system in this condition is effectivelyturned off.

[0007] A variety of tumor immunization strategies have been developed.However, all of these strategies are complex and deviate significantlyfrom the conventional immunization strategies used for infectiousdiseases (Weber J. Tumor, Medscape Anthology 3:2, 2000). One such tumorimmunization strategy involves Theratope®, a Sialyl T_(N) polysaccharidemucin antigen conjugated with keyhole limpet hemocyanine andadministered with Detox® mycobacterium adjuvant and low dosecyclophosphamide (Maclean G D, et al, J Immunother Emphasis TumorImmunol 19(4):309-316, 1996). However, use of this vaccine in patientswith metastatic breast and ovarian cancer has yielded major clinicalresponses in a low percentage of patients. A major response meansgreater than 50% tumor reduction.

[0008] Gene therapy has also been attempted using an adenovirusconstruct as an expression vector for genes expressing Papilloma viruspeptide 16 has been used for immunization or patients with cervicalcancer and has yielded major clinical responses in a low percentage ofpatients (Borysiewickz L K, et al, Lancet 347:1524-1527, 1996).

[0009] Dendritic cell mediated therapy has also been attempted, whereindendritic cells were pulsed with oligopeptide fragments of prostatespecific antigens (PSA). Prostate specific membrane antigen (PSMA) hasbeen used in patients with metastatic prostate cancer with majorclinical responses in a low percentage of patients (Sanda M G, et al,Urology 52:2, 1999; Murphy GP, et al, The prostate. 38:43-78, 1999)

[0010] Additionally, autologous tumors have been used with low dosecyclophosphamide and BCG to immunize cancer patients with malignantmelanoma. However, few clinical responses were reported (Mastrangelo MJ, et al, Seminars in Oncology 23(6):773-781, 1996). Another strategyattempted included using MAGE antigens with a variety of vaccineadjuvants. Again, this has yielded few, if any, responses in patientswith malignant melanoma (personal communication Thierry Boon).

[0011] Several patents to Doyle et al (U.S. Pat. Nos. 5,503,841;5,800,810; 6,060,068; 5,643,565; 5,100,664) disclose methods ofenhancing the immune response in patients using Interleukin 2—(IL-2).This method is disclosed for use in response to infectious diseases andprimarily functions using antigens known to be immunogenic. Limitedapplicability was demonstrated. As disclosed above, the treatment ofcancer is known to require different approaches. To date, treatment withIL-2 has shown minor effects in two cancers, renal cell and malignantmelanoma (response rates less than 20%). It is generally consideredineffective in squamous cell head and neck and cervical cancer and inprostate cancer. Hence, it is not approved for these uses. It wouldtherefore not be within the skill of one in the art to apply the methodof the Doyle et al patents to the use of small peptides in the treatmentof cancer.

[0012] It is important to contrast prevention with known “classic”antigens of complex structure and high molecular weights in healthypatients vs. treatment (generally unsuccessful) with tumor antigens orpeptides (general unsuccessful) in immunosupressed patients (generallyunsuccessful). The first is easy and our current viral vaccines attestto their efficacy. The latter is nearly impossible on a routine basisdespite 30 years of intense effort.

[0013] It is important that this invention relates to, but notexclusively to, immunizing with endogenous peptide processed andpresented by dendritic cells or endogenously administered to anenvironment (lymph node) where dendrtic cells have been prepared and canpresent them to T-cells effectively. This goal is considered by manyimmunologists to be insurmountable. Peptides are much too small to beeffective immunogens, their one half life is short they are oftennonmutated self antigens to which the patient is immunologicallytolerant and gaining a response is tantamount to inducing auto immunity.

[0014] In several of the above strategies, cellular and/or tumoralimmunity to tumor-associated antigens has been induced (Weber J. Tumor,Medscape Anthology 3:2, 2000; Maclean G D, et al, J Immunother EmphasisTumor Immunol 19(4):309-316,1996; Borysiewickz L K, et al, Lancet347:1524-1527, 1996; Sanda M G, et al, Urology 52:2,1999). This isespecially so in association with tumor regression. Nevertheless, thesuccess rate of such treatments is negligible and inconsistent (<30%).

[0015] It would therefore be useful to develop a consistent andeffective method of immunizing cancer patients.

SUMMARY OF THE INVENTION

[0016] In accordance with the present invention there is provided amethod for overcoming immune depression by inducing production of naiveT cells and restoring T-cell immunity. That is the present inventionprovides an immune restoration. The present invention further provides amethod of vaccine immunotherapy including the steps of inducingproduction of naive T cells and exposing the naive T cells to endogenousor exogenous antigens at an appropriate site. Additionally, the presentinvention provides a method for unblocking immunization at a regionallymph node by promoting differentiation and maturation of immaturedendritic cells at a regional lymph node and allowing presentation ofprocessed peptides by resulting mature dendritic cells, thus exposingtumor peptides to T cells to gain immunization of the T cells.Additionally, the present invention provides a method of treating cancerand other persistent lesions by administering an effective amount of anatural cytokine mixture as an adjuvant to endogenous or exogenouslyadministered antigen of the cancer or other persistent lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Other advantages of the present invention are readily appreciatedas the same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

[0018]FIG. 1 is a graph showing a comparison of NCM in different mediautilizing continuous versus pulsed exposure to PHA;

[0019]FIG. 2 is a graph showing the effect of cell concentration withcontinuous exposure to PHA;

[0020]FIG. 3 is a bar graph similar to FIG. 1 with PHA at twice theconcentration (2 micrograms per ml);

[0021]FIG. 4 is a graph of thymidine uptake versus units per ml of IL2relating to splenocytes;

[0022]FIG. 5 is a graph similar to FIG. 2 related to thymocytes;

[0023]FIG. 6 is a graph showing ratio to control versus in vivotreatments for mice with involuted thymuses is treated with IL1, IL2 orIL combinations, NCM, or saline;

[0024]FIG. 7 is a graph also showing a comparison of treatment withrecombinant IL1, IL2, IL1 plus IL2, and NCM;

[0025]FIG. 8 is a graph demonstrating the effect of NCM treatment invivo on splenocyte and thymocyte markers;

[0026]FIG. 9 is a bar graph also demonstrating the effect of NCMtreatment in vivo on splenocyte and thymocyte markers;

[0027]FIG. 10 is a graph demonstrating splenocyte and splenocyteresponses to in vitro media including various recombinant interleukinsor NCM after treatment in vivo with control media or NCM;

[0028]FIG. 11 is a bar graph demonstrating the splenocyte and thymocyteresponses in vitro to media, various interleukins, or NCM in vivo withcontrol media or NCM;

[0029]FIG. 12 demonstrates responses in splenocyte and thymocyte invitro to ConA and PHA after treatment in vivo with control or NCM;

[0030]FIG. 13 demonstrates responses in splenocyte and thymocyte invitro to ConA and PHA after treatment in vivo with control or NCM;

[0031]FIG. 14 is a bar graph showing node size in controls, and cancercontrols or IRX-2(NCM) treated populations with squamous cell head &neck cancer (H&NSCC);

[0032]FIG. 15 shows two bar graphs, one showing T-cell area and thesecond showing density in controls and head and neck squamous cancercontrols and patients treated with NCM(IRX-2);

[0033]FIG. 16 shows two bar graphs showing B-cell area and follicles inthe three treatment groups;

[0034]FIG. 17 shows a comparison of other cells and sinus histocytosisin the three treatment groups; and

[0035]FIG. 18 is a graph showing node B&T and Cancer B&T fit plot.

DESCRIPTION OF THE INVENTION

[0036] Generally, the present invention provides methods for treatingpatients utilizing vaccine immunotherapy wherein the patients are immunesuppressed. By immune suppressed, it is meant that the patient hasreduced cellular immunity and thus impaired capacity to respond to newantigens. More specifically, in blood, T lymphocyte counts are reducedand/or function of these cells is impaired, as shown, e.g. by PHAproliferation assay.

[0037] T lymphocytopenia (low T cell levels in blood) is a diagnosticcharacteristic of cellular immune deficiency; impaired function ofexisting thymphocytes is the other characteristic. There is no generallyaccepted (clinically approved) way to treat T lymphocytopenia. Bonemarrow transplants (± thymus transplants) have been used in cases ofsevere combined immunodeficiency (SCID—congenital, irradiation orchemotherapy induced). Recombinant IL2 has been tried in AIDS with someeffect by much toxicity.

[0038] There are two ways to make new T cells to attempt to correct Tlymphocytopenia. One way, as in rIL-2 therapy, expands T cells alreadyin the periphery, i.e., memory T cells (CD₄₅RO) (blood, lymph node andspleen). The other involves processing in the thymus of new T cells frombone marrow - derived precursors. This happens naturally in children butnot in adults. These new cells are called recent “thymic émigrés” andhave the surface marker of “naïve” T cells i.e., CD₄₅RA. NCM therapy(plus Thymosin a₁) results in the production of these new T cells aswell as expanding preexisting memory T cells.

[0039] More specifically, the present invention utilizes new discoveriesrelating to immunization to provide an immune response to antigens whichis either endogenously or exogenously administered. Such antigens in thepast may have been believed to be immunogenic while others used in thepresent invention may have been thought previously to benon-immunogenic. Examples of such antigens are EADPTGHSY (melanoma) fromMAGE-1 protein, EVDPIGHLY (lung carcinoma) from MAGE-3, EVDPIGHLY (lungcarcinoma) from MAGE-3, and many others. (See Bellone, et al, ImmunologyToday, Vol 20, No.10, p 457-462,1999.)

[0040] The present invention utilizes several general newly derivedmethod steps for obtaining immunization in subjects where suchimmunization was previously thought to be impossible. More specifically,the present invention provides a method for overcoming immune depressionby inducing production of naive T cells. The term “naive” T cells ismeant to mean newly produced T cells, even in adults, wherein these Tcells have not yet been exposed to antigen. Such T cells at this stageare non-specific yet capable of becoming specific upon presentation by amature dendritic cell having antigen, such as tumor peptides, exposedthereon. Thus, the present invention replenishes or generates new Tcells. This is generally accomplished by administering a naturalcytokine mixture (NCM). The NCM includes IL1, IL2, IL6, IL8, IL10, IL12,δIFN, TNFα and G- and GM-CSF. The amount and proportions of theseconstituents are detailed below. Preferably, about 150-600 units of IL2are contained in the NCM.

[0041] Preferably, the NCM is injected around lymphatics that drain intolymph nodes regional to a lesion, such as a tumor or other persistentlesions being treated. Perilymphatic administration into the lymphaticswhich drain into the lymph nodes, regional to the lesion, such as acancer, is critical. Peritumoral injection has been associated withlittle response, even progression and is thus contraindicated. A ten(10) day injection scheme is optimal and a twenty (20) day injectionprotocol, while effective clinically, tends to reduce the TH1 responseand shift towards a less desirable TH2 response as measured by lymphoidinfiltration into the cancer. Bilateral injections are effective. Whereradical neck dissection has occurred, contralaterial injection iseffective.

[0042] It is preferable to block endogenous suppression of T cells, suchas caused by various cancer lesions. Blocking is effected by thecodelivery of low dose cyclophosphamide and a non-steroidalanti-inflammatory drug (NSAID). The NSAID of choice is indomethacin.While indomethacin is the most effective NSAID, it is also arguably themost toxic. Celebrex® and Vioxx®, Cox II NSAIDS, are less effective.Vioxx® can be more toxic, causing gastritis in many patients. Ibuprophenwas effective but the histological responses were characteristic of aTH2 rather than TH1 mediated response, this being less desirable. Sideeffects of NSAIDS are to be aggressively treated with proton inhibitorsand a prostaglandin E analog. Zinc and multi-vitamins are useful agentsto help restore T cell immunity. Applicants have found that treatmentwith contrasuppression and zinc without the NCM is ineffective.

[0043] In summary, the minimum regimen is perilymphatic treatment withthe NCM combined with contrasuppression using cyclophosphamide and anNSAID. The alternative regimen is the previously mentioned regimenfurther including zinc and vitamins, possibly including the addition ofselenium. Preferable dosing of Zinc is 50 to 75 mg. A standardmultivitamin can be administered. The zinc can be an availablegluconate.

[0044] In order to maximize clinical response and for the greatestincrease in survival rate, the degree and type of lymphocyteinfiltration is important. Lymphocyte: granulocyte or macrophageinfiltration of a 90:10 ratio is optimal. T and/or B cell infiltrationpreferably is diffuse and intense and not peripheral. Light infiltrationof less than 20% is not associate with a robust clinical response. Tumorreduction and fragmentation in the histological samples is preferred inreflecting a good response. Lymph node changes key to good responseinvolve at least five (5) aspects. Lymph node enlargement and not justreversal of tumor induced reduction of size but overall increase in sizecompared to normal is preferred. Increased T and B cell areas indicatean immunization. Sinus histocytosis (SH) is believed to be theaccumulation of immature dendritic cells which have ingested andprocessed tumor antigens but are unable to mature and present thesetumor peptides to naive T cells capable of stimulating TH1 and TH2effective cells which lead to cytotoxin T cell and B cells. Reversal ofSH is preferred

[0045] Thus, the present invention provides for unblocking immunizationat a regional lymph node by promoting differentiation and maturation ofimmature dendritic cells in a regional lymph node and thus allowingpresentation by resulting mature dendritic cells of small peptides,generally nine amino acids in length to T cells to gain immunization ofthe T cells. Additionally, induction of mature dendritic cells isrequired. Finally, mobilization of peripheral blood T-lymphocytes inT-lymphocytopoenic patients in the presence of induction of naive Tcells capable of responding to dendritic cells presenting endogenoustumor peptides is desired. (See Sprent, et al, Science, Vol 293, July13, 2001, pgs 245-248).

[0046] In view of the above, the key mechanistic features of the presentinvention are the in vivo maturation of dendritic cells resulting ineffective peptide antigen presentation. Based on the examples presentedbelow, increases in CD45 RA positive naïve uncommitted T cells have beenfound. With antigen, this leads to T and B cell clonal expansion,creating immunity in the patient. The resulting infiltration into tumorsby hematogenous spread leads to robust tumor destruction. The result, asfound in the data below, is increased survival due to immunologicmemory. (See Sprent et al, cited above).

[0047] It is predicted logically that exogenously provided synthetic orextracted tumor peptides (See Bellone, et al, cited above) can bedelivered into the pre-primed or co-primed regional or distal lymph nodeand yield tumor antigen specific T cells, with or without B cells. Threeexamples are set forth below. In view of the above, it can be concludedthat the action of NCM plus other agents is useful as for any tumorantigens (synthetic and endogenous, peptides and proteins). Many ofthese peptides are not normally immunogenic and only when presented by amatured, activated dendritic cell, will they be effective in immunizingnaïve T cells. Thus, the appearance of an immune T cell means, de facto,that a dendritic cell has been made or allowed to work properly. Also defacto, dendritic cell activation and maturation is to be considered akey factor in cancer immunodeficiency as well as the well-known defectsin T cells such as a decreased number and function with anergy andpresumed apoptosis.

[0048] Referring more specifically to the protocol and medicantdelivered in accordance with the present invention, the inventionutilizes the natural cytokine mixture (NCM) to immunize patients, suchas cancer patients, as well as patients with other lesions or antigenproducing disease conditions.

[0049] More specifically, the present invention utilizes a method ofenhancing the immune response of cancer patients to a cancer byadministering an effective amount of a composition containing thereinthe NCM and a tumor-associated antigen, the NCM acting as an adjuvant toproduce the immune response. The tumor associated antigen can be eitheran endogenously processed tumor peptide preparation resident in regionalnodes of patients with cancer or in conjunction with an exogenouslyadministered tumor antigen preparation in or near these nodes. Tumorpeptides, as well as antigens, are included herein even though peptidesare not expected to be immunogenic where tumor associated proteinantigens would more likely be more so since they are complete.

[0050] In the preferred embodiment, the composition of the presentinvention involves the administration of the NCM plus a tumor associatedor specific antigen, as defined below with low doses ofcyclophosphamide, a cyclooxygenase inhibitor, and other similarcompounds which have been shown to further increase the effects of thecomposition of the present invention.

[0051] To clarify and further define the above, the followingdefinitions are provided. By “adjuvant” it is meant a composition withthe ability to enhance the immune response to a particular antigen. Tobe effective, an adjuvant must be delivered at or near the site ofantigen. Such ability is manifested by a significant increase in immunemediated protection. Enhancement of immunity is typically manifested bya significant increase (usually greater than 10 fold) in the titer ofantibody raised to the antigen. Enhancement of cellular immunity can bemeasured by a positive skin test, cytotoxic T-cell assay, ELISPOT assayfor δIFN or IL-2, or T-cell infiltration into the tumor (as describedbelow).

[0052] By “tumor associated antigen”, it is meant an analogous proteinor peptide (which were previously shown to work by pulsing of dendriticcell ex vivo) or other equivalent antigen. This can include, but is notlimited to PSMA peptides, MAGE peptides (Sahin U, et al, Curr OpinImmunol 9:709-715, 1997; Wang RF, et al, Immunologic Reviews 170:85-100,1999), Papilloma virus peptides (E6 and E7), MAGE fragments, NY ESO-1 orother similar antigens. Previously, these antigens were not consideredto be effective in treating patients based either on their size, i.e.they are too small or that they were previously thought to not have theimmunogenic properties (i.e., self antigens).

[0053] NCM, a non-recombinant cytokine mixture, is defined as set forthin U.S. Pat. Nos. 5,632,983 and 5,698,194. Briefly, NCM is prepared inthe continuous presence of a 4-aminoquinolone antibiotic and with thecontinuous or pulsed presence of a mitogen which in the preferredembodiment is PHA.

[0054] According to the present invention, there is provided a partiallycharacterized NCM that has been previously shown to be effective inpromoting T cell development and function in aged, immunosuppressedmice. Upon administering this NCM to immunosuppressed patients with headand neck cancer, it is demonstrated in this application for the firsttime that the mobilization of T lymphocytes in the blood of cancerpatients treated with the NCM produces an increase in immature, naive Tcells bearing both CD2 and CD45 RA. This is one of the firstdemonstrations that adult humans can generate naïve T cells. Previousreferences: Mackall et al, (New England Journal of Medicine (1995), Vol.332, pp. 143-149); and a review by Mackall (Stem Cells 2000, Vol. 18.pp. 10-18) discusses the inability to generate new T cells in adults butnot children, and discusses the problem of trying to replenish T cellsfollowing cancer chemotherapy and/or radiotherapy. In general there isthe dogma that new T cells are not generated in the adult human.However, following bone marrow transplantation for intense chemotherapy,there has been evidence that new T cells can be generated in the adult.No molecular therapy to date has been able to achieve this, althoughincrease in lymphocytes counts have been achieved with prolonged andintense therapy with recombinant interleukin-2 in patients infected byHIV. These have not been clearly demonstrated to be thymus derived Tcells and are presumably an expansion of pre-existing peripheral Tcells.

[0055] Previously, Cortesina et al. employed a natural IL-2,perilymphatically in patients with head and neck cancer and inducedseveral tumor regressions (Cortesina G, et al, Cancer 62:2482-2485,1988) with some tumor infiltration with leukocytes (Valente G, et al,Modern Pathol 3(6):702-708, 1990). Untreatable recurrences occurred andthe response was termed non-specific and without memory and thusnonimmunologic (Cortesina G, et al, Br J Cancer 69:572-577, 1994). Therepeated attempts to confirm the initial observations with recombinantIL-2 were substantially unsuccessful (Hadden J W, Int'l JImmunopharmacol 11/12:629-644, 1997).

[0056] The method of the present invention involves using NCM with localperilymphatic injections or other injections that are known to those ofskill in the art to provide sufficient localization of the immunotherapycompound. In the preferred embodiment, the injections take place in theneck, but can be applied in other locations as required by the diseaseto be treated. This treatment induced clinical regressions in a highpercentage of patients who also showed improved, recurrence freesurvival (Hadden J W, et al, Arch Otolaryngol Head Neck Surg.120:395-403, 1994; Meneses A, et al, Arch Pathol Lab Med 122:447-454,1998; Barrera J, et al, Arch Otolaryngol Head Neck Surg 126:345-351,2000; Whiteside, et al, Cancer Res. 53:564-5662, 1993). Whiteside, et al(Cancer Res. 53:5654-5662, 1993) observed that in head and neck cancer,tumoral injection of recombinant interleukin-2 produced a T celllymphocyte infiltrate, but without significant clinical responses.Peritumoral injection of Multikine (Celsci Website) (in combination withperilymphatic injection in up to 150 patients resulted in significanttumor responses, i.e. greater than 50% tumor reduction in only 11patients, making their response rate less than 10% in contrast to thehigh degree of response observed in the present studies, 40%. Inaddition, they noted 50% non-responders where Applicants have observedonly 20%.

[0057] Applicants, have observed that peritumoral and intratumoralinjection can be associated with progression of disease even in patientswho initially have had a positive response to the NCM protocol, thusundoing its benefit. Peritumoral injection is thus contraindicated andis excluded as part of the present invention. This has led Applicants tothe interpretation that the tumor is not the site of immunization andthe present application presents documentation that the regional lymphnode is the site of immunization. Then, unpublished analysis of regionallymph nodes revealed data which indicated that the regional lymph nodeis the site of immunization to postulated tumor antigens (FIGS. 14-18).With the identification of a number of different tumor antigens, it hasbeen a conundrum over the last decade that given the presence of suchantigens, they have not been employed effectively in immunizationprotocols. Sporadic positive examples have been reported, but in themain, the data are negative. The problem of antigen presentation hasbeen focused on in the last decade and the dendritic cell has emerged asa critical player in the presentation of small peptides derived fromtumors. See DeLaugh and Lotts, Current Opinion In Immunology, 2000, Vol.12, pp.583-588; Banchereau et al, Annual Reviews of Immunology, (2000),Vol. 18, pp. 767-811; also Albert et al, Nature, Vol. 392, pp.86-89(1998).

[0058] In brief, in order for tumor antigens to be properly antigenic,they must arrive from an apoptotic rather than a necrotic tumor cell(Albert, Nature, 39 2:86-87, 1997). They need to be captured by immaturedendritic cells that have the morphology of large histocytes. Theseimmature dendritic cells process antigen (endocytosis, phagocytosis anddigestion) and evolve into mature dendritic cells which display peptidefragments (generally nine amino acids) of the digested antigen in theMHC groove for presentation to T cells. T cells, in order to respond,must have antigen presented to them in the MHC groove plus variousco-stimulatory signals. References: Banchereau and DeLaugh.

[0059] Investigators, such as Murphy et al, 1999, have utilizeddendritic cells generated in culture and then pulsed with tumor antigensand have achieved a small degree of success in immunizing patientsagainst prostate specific membrane antigen peptides. Unfortunately, thisapproach of pulsing dendritic cells is cumbersome and has been ratherinefficient. In the present invention, Applicants have shown that thecells present in the lymph node sinuses, which accumulate in cancer, arecells of the lineage of dendritic cells and that following the in vivotreatment with the NCM protocol, these cells disappear and antigenultimately then becomes immunogenic for T cells. They are able then torespond to the tumor. So a critical aspect of this invention is beingable to generate a microenvironment in the regional lymph node whichallows effective antigen processing and presentation. The immunizationwhich derives results in T cells able to traffic to the lesion anddestroy tumors is de facto demonstration of adequate antigen processingby dendritic cells. Additionally, none of the patients treated with NCMdeveloped distant metastasis which is expected in up to 15% clinicallyand up to 50% pathologically. This indicates that a systemic immunityrather than merely a local immunity has been induced by the treatment.This is a drastic improvement over the compositions in the prior art,because the prior art compositions, at best, were inconsistentlyeffective against metastatic disease. The ability of the composition ofthe present invention to create systemic immunity allows more effectiveand efficient treatment of a patient. Further, the magnitude of systemicresponse enables an individual to be administered smaller doses withoutlimiting the effectiveness of the treatment and without toxicity.

[0060] The literature (Hadden J W, Int'l J Immunopharmacol11/12:629-644, 1997; Hadden J W. Immunology and immunotherapy of breastcancer: An update: Int'l J Immunopharmacol 21:79-101, 1999) hasindicated that for both SCC and adenocarcinomas, the two major types ofcancer, regional lymph nodes reflect abnormalities related to the tumor,including sinus histocytosis, lymphoid depletion and often the presenceof anergic tumor associated lymphocytes (capable of reacting to tumorcells with ex vivo expansion and recovery using IL-2). Then, withmetastases, lymphoid depletion and depressed function occur.Additionally, uninvolved cervical lymph nodes of such patients haveshown a reduction in average size and an increase in sinus histocytosisassociated with head and neck cancers. (See FIGS. 14-17).

[0061] Specifically relating to the composition, the composition of thepresent invention involves the natural cytokine mixture plus eitherendogenous or exogenous tumor associated antigen. Additionally, lowdoses of cyclophosphamide, cyclooxygenase inhibitors, zinc, and othersimilar compounds have been shown to further increase the effects of thecomposition of the present invention.

[0062] Immunization for treatment of patients with cellular immunedeficiencies associated with cancer, HIV infection, aging, renaltransplants and other such deficiencies can be achieved with thecomposition of the present invention.

[0063] Administration and protocols for treatment as follows:

[0064] Delivery of gene Products/Synthetic Antigens with:

[0065] The compounds of the present invention (including NCM), andexogenous antigens are administered and dosed to achieve optimalimmunization, taking into account the clinical condition of theindividual patient, the site and method of administration, scheduling ofadministration, patient age, sex, body weight. The pharmaceutically“effective amount” for purposes herein is thus determined by suchconsiderations as are known in the art. The amount must be effective toachieve immunization including but not limited to improved tumorreduction, fragmentation and infiltration, survival rate or more rapidrecovery, or improvement or elimination of symptoms.

[0066] In the method of the present invention, the compounds of thepresent invention can be administered in various ways. It should benoted that they can be administered as the compound or aspharmaceutically acceptable salt and can be administered alone or as anactive ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The compounds can beadministered intra or subcutaneously, or peri or intralymphatically,intranodally or intrasplenically or intramuscularly, intraperitoneally,and intrathorasically. Implants of the compounds can also be useful. Thepatient being treated is a warm-blooded animal and, in particular,mammals including man. The pharmaceutically acceptable carriers,diluents, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

[0067] The doses can be single doses or multiple doses over a period ofseveral days.

[0068] When administering the compound of the present invention, it isgenerally formulated in a unit dosage injectable form (solution,suspension, emulsion). The pharmaceutical formulations suitable forinjection include sterile aqueous solutions or dispersions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

[0069] Proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it is desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

[0070] Peptides may be polymerized or conjugated to carriers such ashuman serum albumen as is well known in the art.

[0071] Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

[0072] A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, additives, and diluents; orthe compounds utilized in the present invention can be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Examples of delivery systems useful in the presentinvention include: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other such implants, delivery systems,and modules are well known to those skilled in the art.

[0073] The foregoing provides a protocol for using NCM as an adjuvant toimmunize cancer patients against tumor antigens, either autologous or asdefined proteins or peptides. The antigen preparations to be used: InCancer: 1) PSMA peptides (9) - obtained commercially Prostate 2) MAGE 1& 3 & MAGE fragments & NY ESO-1 Melanoma, obtained from the Ludwig Inst.Of Immunol. H&NSCC 3) Papilloma virus E6 & E7 obtained commerciallyCervical SCC

[0074] The commercially route of antigen administration ispreferentially the neck because it is accessible and it contains >30% ofthe bodies lymph nodes and systemic immunity can be envisioned toresult.

[0075] Low Dose Cyclosphosphamide:

[0076] Low dose CY has been used to augment cellular immunity anddecrease suppression by lymphocytes in mice and patients with cancer(Berd D., Progress in Clin Biol Res 288:449-458, 1989; Berd D, et al,Cancer Research 47:3317-3321, 1987) and it has been employed ineffective immunotherapy of cancer patients (Weber J., Medscape Anthology3:2, 2000; Murphy G P, Tjoa B A, Simmons S J. The prostate. 38:43-78,1999; Hadden J W, et al, Arch Otolaryngol Head Neck Surg.120:395-403,1994).

[0077] Zinc:

[0078] Zinc deficiency is associated with improved cellular immunity andtreatment with zinc is immunorestorative in mice (Hadden JW., Int'l JImmunopharmacol 17:696-701, 1995; Saha A., et al. Int'l JImmunopharmacol 17:729-734,1995).

[0079] A cyclooxygenase inhibitor (COXi) like indomethacin: Cancersproduce prostaglandins and induce host macrophage production ofprostaglandins (Hadden J W. The immunopharmacology of head and neckcancer: An update. Int'l J Immunopharmacol 11/12:629-644,1997). Sinceprostaglandins are known to be immunosuppressive for T cells, inhibitionof PG synthesis with cyclooxygenase inhibitors is appropriate.

[0080] Recombinant Protein Purification

[0081] Marshak et al, “Strategies for Protein Purification andCharacterization. A laboratory course manual.” CSHL Press, 1996.

[0082] Dose and Frequency of Antigens

[0083] 1-1000 μg, preferably 10-500; form—soluble (partially polymerizedor conjugated to carrier, if necessary)

[0084] Schedule: Day 1, Day 12, Day 21

[0085] (Pre-Rx) Day 12, Day 21, Day 31

[0086] Site of injection: local injection, ie. neck injections

[0087] Expected Responses: Tumor reduction

[0088] Tumor pathological changes (reduction, fragmentation, lymphoidinfiltration)

[0089] Humoral immunity to antigen (RAI or ELISA)

[0090] Cellular immunity to antigen (intracutaneous skin test in vitrolymphocyte proliferation, of ELISPOT ASSAY)

[0091] Keep in mind that oligopeptides like PSMA, MAGE fragments, E6, E7peptides would be poorly immunogenic even pulsing on to dendritic cells.Thus effective immunization would not be expected to occur. Even witheffective immunization, tumor regression would be considered surprisingby this method, particularly at a distance as with prostate and cervix.Regression of metastastic disease is always a surprising event withimmunotherapy. Degree and frequency of clinical responses are a factorin the effectiveness and thus the novelty of this approach.

[0092] Diagnostic skin tests are another way to guide us to moreeffective immunization. Patients can be pretreated with IRX-2 (NCM) toinduce better responses (increase NCM and PHA skin tests and lymphocytecounts and reversal of lymph node abnormalities).

[0093] This creates an Adjuvant strategy

[0094] Combining immunorestoration and adjuvancy

[0095] Making peptides and proteins immunogenic

[0096] Getting the degree of immune response to effect tumor regressionat a distance.

[0097] It can extend to all forms of tumor antigens and haptensincluding peptides and/or carbohydrates.

[0098] It can extend to areas of applicability as in AIDS virus vaccinein HIV+ patients; other difficult to manage situations; renaltransplants, aged, etc.

[0099] Patients will be skin tested for one or more tumor peptide priorto consideration of the protocol, 100 pg of one or more tumor peptideswill be perilymphatically administered in the neck with NCM using theNCM protocol as discussed below on day 1 and 10 of the NCM series. Thecombination will be repeated on day 21. In addition to tumor responseand histology, immune reaction to the peptides will be monitored byrepeat skin test or by other means known in the art.

EXAMPLE 1

[0100] All steps relating to cell culture are performed under sterileconditions. General methods of cellular immunology not described hereinare performed as described in general references for Cellular immunologytechniques such as Mishell and Shiigi (Selected Methods in CellularImmunology, 1981) and as are known in the art.

[0101] Preparation of Natural Cytokine Mixture (NCM)

[0102] The buffy coat white cells of human blood from multipleHIV-negative hepatitis virus-negative donors is collected. In analternative embodiment, animals could be the cell source for veterinaryuses. The cells from the donors are pooled and layered on ficoll hypaquegradients (Pharmacia) to yield lymphocytes free of neutrophils anderythrocytes. Alternative methods could be used that would result in thesame starting lymphocyte population as are known in the art.

[0103] The lymphocytes are washed and distributed in X vivo-10 media(Whittaker Bioproducts) to surface activated cell culture flasks forselection of cell subsets MICROCELLECTOR.TM. T-25 Cell Culture Flasks)in which are immobilized stimulants, i.e. mitogens like PHA. In one setof experiments, X vivo-15 and X vivo-20 media were used as indicated.The immobilization process for the stimulants is as described by themanufacturer for immobilizing various substances for panning procedures,i.e. separating cells, in the flasks. Alternatively, the lymphocytes areexposed to stimulants e.g. PHA for 2-4 hours then washed three times.

[0104] The cells are incubated for 24-48 hours in X vivo-10 media with80 μg/ml ciprofloxacin (Miles Lab) at 370 in a CO₂/air incubator.Alternatively, RPMI 1640 media could be used (Webb et al. 1973).Generally the HSA is used at 0.1 to 0.5% (weight by volume). Followingincubation the supernatants are poured off and collected. Human serumalbumin (HSA) may be added to stabilize further the interleukins ifHSA-free media is used for generations. The supernatants are stored at4° C. to −70°

[0105] Characterization of Supernatants

[0106] The pooled supernatants are characterized by measuring thecytokine content by bioassay for IL-2 and ELISAs for the remaininginterleukins IL-1-IL-15, CSFs, TNFs, and IFNs. Sterility is tested byculture in thioglycolate broth and endotoxin measured by limulus lysateassay as is known in the art.

[0107] Standardization of supernatant for cytokine content:

[0108] Each supernatant is standardized either by concentration oramount administered so that comparisons can be made.

[0109] Removal of contaminants from supernatant:

[0110] DNA and virus exclusion, if used, employ such techniques asultrafiltration, column chromatography, virus retentive filters, ethanolfractionation, polyethylene glycol/bentonite precipitation, gammairradiation, and/or solvent/detergent treatment as has been used forintravenous gamma globulin and monoclonal antibodies (e.g. IGIV NewsUpdate brochure).

[0111] Model

[0112] The model of hydrocortisone induced thymic involution in agedmice was used unless otherwise indicated (Hadden J W, et al, Int'l JImmunopharmacol 17:821-828. 1995).

[0113] Laboratory Animals

[0114] Female BALB/c (Life Science, St. Petersburg, Fla.) aged retiredbreeder mice (8-9 months) whose thymuses had begun to involute wereemployed in in vivo tests. Mice were weight matched and randomly pooledin groups of five. Animals were fed standard laboratory diets withdrinking water ad lib. All mice, with exception of a control group, weretreated intraperitoneally (i.p.) with hydrocortisone (5 mg/mouse in 0.1ml 0.9% sodium chloride) for two consecutive days to induce a chemicalthymectomy and reduction of spleen weight.

[0115] Hydrocortisone-treated adult mice show acute thymic involution(less than 30% of control) and reduction in spleen size (less than 80%of control) at two days with progressive recovery to 10 days.

[0116] Experimental Design

[0117] Each treatment group had five (5) animals and each experiment wasrepeated 2-5 times. Treatment was initiated intraperitoneally (i.p.) onDay 3 and continued once per day for a total of five (5) days. Treatmentgroups were injected with one of the following in vivo treatments asindicated in the text:

[0118] 1. pyrogen free saline (controls);

[0119] 2. recombinant interleukin-1 (rIL-1; 4 ng);

[0120] 3. recombinant interleukin-2 (rIL-2; 50 units);

[0121] 4. rIL-1+rIL-2 (4 ng+50 units, respectively)

[0122] 5. natural cytokine mixture (NCM; 50 units IL-2 equivalence)

[0123] On day 8, the mice were weighed, sacrificed by cervicaldislocation, and their spleens and thymuses removed and weighed. Theorgans were minced, the residual erythrocytes were lysed using ammoniumchloride (Mishell and Shiigi 1981), and the cells counted.

[0124] The proliferative response of the cells to various substances wasthen determined. A sample of cells was prepared for cell culture at 37°C., 5% CO₂ in RPMI 1640 medium with 5% fetal bovine serum, penicillin(100 U/ml), streptomycin (100 μg/ml) and 2-mercaptoethanol (2×10⁻⁵ M).The cells were plated in 0.2 ml microwell plates in quadruplicate at aconcentration of 1.5×10⁶/ml and incubated for 72 hours with one of thefollowing as indicated in the text:

[0125] 1. control diluent (complete RPMI 1640 medium);

[0126] 2. rIL-1 (1 ng/ml);

[0127] 3. rIL-2 (2 Units/ml);

[0128] 4. NCM (2 Units/ml of IL-2 equivalence)

[0129] 5. concanavalin A (Con A; 1.5 μg/ml)

[0130] 6. phytohemagglutinin (PHA; 0.5μg/ml)

[0131] The culture was terminated to measure DNA synthesis, thus cellproliferation, with an 18 hours pulse of tritiated thymidine(3H-Thymidine; New England Nuclear, Boston, Mass.; specific activity 6.7Ci/mM), harvested with a multiple automatic sample harvester andprocessed for liquid scintillation counting. Marker studies were alsoperformed as described by, Hadden et al. (1992). The results wereexpressed as arithmetic mean of cpm from three samples for each animal.In order to simplify the representation of data obtained with differentanimals, the results with the different animals were pooled andcalculated together and in some cases are expressed as ratio to controland others as means+brackets for standard error of the mean (SEM).

[0132] Statistical Analysis

[0133] Student's T test was used to analyze data as appropriate.

[0134] Results:

[0135] The objective was to find a way to stimulate lymphocytes toproduce high levels of interleukin-2 in the absence of serum and in away which did not yield significant quantities of PHA in thesupernatant. To do this, the PHA was immobilized on surface activatedcell culture flasks for selection of cell subsets (AISMICROCELLECTOR.TM. T-25 plates) as described in the manufacturer'sinstructions for “panning” cell separation or pulsed into the cellsfollowed by washing (pulse technique).

[0136] Media employed in these experiments was X vivo-10 (Whittaker) andis approved for administration to humans by the U.S. Food and DrugAdministration for interleukin-2-lymphokine activated killer (LAK) cellprotocols. Serum-free media capable of supporting human lymphocyteproliferation like minimal essential media (MEM) or RPMI-1640 (Sigma)could also be used.

[0137] Initial experiments indicated that PHA (HA-16, Murex DiagnosticsLtd., Dartford, U.K.) could be immobilized by the technique described bythe manufacturer and that under appropriate optimal conditions of cellnumber of 7.5-15×10⁶/ml, time of exposure of 24 hours-48 hours, and PHAconcentration of 25 or 50 μg/ml a high yield of interleukin-2 in theserum-free supernatant could be obtained. The yield was superior to thepulse technique employing brief exposures to PHA (NI) followed bywashing and subsequent culture with ciprofloxacin (NIM) in serum-freemedia (Table 1). TABLE I IL content of supernatant/ml PHA brief exposure(NI) 2-20 units PHA brief exposure 8-140 units & ciprofloxacin (NIM) (80μg/ml) PHA flask immobilization 100-353 units & ciprofloxacin (80 μg/ml)

[0138] IL-2 content was measured in the supernatant using the CTLL IL-2dependent cell line by the methods described by Gillis et al. (1978).IL-2 was quantitated in international units against a known standardcontaining 640 units (Pharmacia AB).

[0139] The cell free supernatants from flasks incubated without cellswere tested on human lymphocytes to determine if residual PHA waspresent in sufficient quantities to produce a proliferative response.Any residual PHA greater than 0.01 μg/ml would give such a response. Inthe absence of cells, small amounts of PHA were observed in thesupernatant at 40-48 hours; however, when PHA (25 μg/ml) was used foronly 24 hours, these levels were negligible. 24 hours incubation wasthus considered optimal. A comparison of X vivo-10, X vivo-15 and Xvivo-20 (Whittaker) and MEM in the present invention was undertaken andshown in FIGS. 1-3. X vivo-10 and X vivo-15; are approved foradministration to humans by the U.S. Food and Drug Administration forinterleukin-2-lymphokine activated killer (LAK) cell protocols.Generation of NCM was compared in different media utilizing continuousvs. pulsed exposure to PHA at 1 μg/ml (FIG. 1). The effect of cellconcentration was explored with continuous exposure to PHA at 1 μg/ml(FIG. 2) and PHA at 2 μg/ml (FIG. 3). The optimal combination of thesefactors was found to be continuous exposure by immobilization inX-vivo-10 at cell concentrations of 2.5 or 5.0×10⁶/ml with PHA at 2μg/ml or at 5×10⁶ cells/ml with PHA at 1 μg/ml. Because the per cellyield is most efficient at 2.5×10⁶ cell/ml, that concentration with PHAat 2 μg/ml is chosen as the optimal.

[0140] Preliminary experiments, in tubes rather than flasks, wereperformed to determine the parameters for ciprofloxacin and two other4-aminoquinolone antibiotics (Norfloxacin and Ofloxacin) to enhancecytokine production from human leukocytes following exposure to PHA.Table III shows that 80 μl/ml of each of these 4-aminoquinoloneantibiotics enhanced production of IL-1, IL-2, IL-6, IFN.gamma.,TNF.alpha., and G-CSF. IL-8 production was maximal. IL-3, IL-4, and IL-7were undetectable under these circumstances in all supernatants. Theseresults indicate that under these serum free conditions all4-aminoquinolones tested at 80 pg/ml enhanced PHA induced cytokineproduction under serum-free conditions. TABLE II PHA CiprofloxacinNorfloxacin Ofloxacin Alone & PHA & PHA & PHA IL-1-β   81 1080 783 810IL-2 ND 120 32 82 IL-6 1665 >3000 >3000 >3000 IL-818000 >18000 >18000 >18000 IFN.gamma.   ND 750 210 380 TNF α   54 19351500 4000 GM-CSF 114 4.5 4.5 72 G-CSF 41 555 800 630

[0141] It was also determined that a monoclonal antibody, OKT-3, (Ortho)which induces T lymphocytes to proliferate and produce interleukinscould be employed as a stimulant under these conditions. Table III showsthat OKT-3 induced cytokines similar to those induced by PHA plusciprofloxacin with cells incubated in flasks as set forth in Example 1.IL-3,4,5 and 7 were not detected with either set of stimulants. OKT-3produced a small additive effect for several ILs when joined with PHAand ciprofloxacin (CIPRO). TABLE III CIPRO OKT-3 + CIPRO + PHA + PHAOKT-3 IL-1-β   1080 1530 1125 IL-2 120 340 ND IFN gamma.   750 466011280 IL-6 >3000 >3000 1980 IL-8 >18000 >18000 >18000 TNF alpha   19352700 2500 GM-CSF 4.5 12 75 G-CSF 555 375 ND

[0142] Units of interleukins other than IL2 are pg/ml and for IL2international units/ml. ND not done.

[0143] In order to show the superiority of the NCM over rIL-2 in vitro,mouse splenocytes and thymocytes were cultured with MEM and rIL-2 atcomparable levels of IL2 as determined by bioassay and DNA synthesismeasured by tritiated thymidine incorporation. NCM induces greaterproliferation of splenocytes (FIG. 4) and thymocytes (FIG. 5) then rIL-2based on IL2 content.

[0144] In a series of experiments as set forth in FIGS. 6 and 7, micewith involuted thymuses were treated in vivo with rIL-1, rIL-2,combinations of these factors, NCM or saline (controls). The spleens andthymuses were removed, the cells tested for cell proliferation responsesagainst the interleukins (IL-1, IL-2 ), NCM and the mitogen ConA. Theresults are expressed as ratio to the saline treated control. In vivotreatment with rIL-1, rIL-2, and their combination (rIL-1 and rIL-2 )had no significant effect to increase proliferative responses ofsplenocytes (FIG. 6) or of thymocytes (FIG. 7) to in vitro stimulationwith IL-1, IL-2, NCM or ConA. NCM treatment in vivo augmentedsignificantly both splenocytes and thymocytes to all four stimuli. Theseresults are consistent with an enhanced sensitivity of these cells tostimulation and/or an increase in the number of responsive cells.

[0145]FIGS. 8 and 9 demonstrate the effect of NCM treatment in vivo onsplenocyte and thymocyte markers. Non-mature T-cells are indicated by −−and may represent T lymphocyte precursors particularly in the thymus.NCM increased proportionately this population in spleen and thymus.Immature T-cells are indicated by ++ and this population isproportionately decreased in thymus by NCM treatment. Mature T-cells areindicated by CD4+ and CD8+. NCM increased the proportions of matureT-cells in thymus and their number in spleen. These results areconsistent with an effect of NCM to increase T cell precursors and topromote their development to mature T cells in thymus.

[0146]FIGS. 10 and 11 demonstrate the splenocyte and thymocyte responsesin vitro to media (RPMI), rIL-1 (IL1), rIL-2 (IL₂), or NCM aftertreatment in vivo with control media or NCM in the hydrocortisone model.The mice were treated as described hereinabove. These data demonstratethat NCM augments background splenocyte responses, splenocyte responsesto IL-1 and IL-2, but not NCM and background thymocyte responses andthymocyte responses to IL-1, IL-2, and NCM.

[0147]FIGS. 12 and 13 demonstrate the splenocyte and thymocyte responsesin vitro to ConA and PHA after treatment in vivo with control media orNCM. The mice were treated as described hereinabove.

[0148] The in vitro studies demonstrate the superiority of NCM overrIL-2 at equivalent doses in sensitizing splenocytes and thymocytes toproliferation signals. The effects on thymocytes reflect promotion ofdifferentiation as well. The NCM composition, but not rIL-1, rIL-2, northeir combination, potently promotes in vivo T lymphocyte function (ILresponses) and development (mitogen responses and cell markers) which istherapeutically relevant in any therapeutic measures requiringstimulation of the immune system or restoring even partial functioningof a damaged or defective immune system. For example chemotherapeuticagents can damage cells, including T lymphocytes, involved in the immuneresponse. The present invention by stimulating the T lymphocytefunctioning and development can restore, either partially or entirely,this feature of the immune system if damaged.

EXAMPLE 2

[0149] There is shown that local perilymphatic injections in the neckhaving NCM plus low dose cyclophosphamide, indomethacin, and zinc andinduced clinical regressions in a high percentage of patients withsquamous cell head and neck cancer (H&NSCC) (Hadden J W, et al., ArchOtolaryngol Head Neck Surg. 120:395-403, 1994; Meneses A, et al., ArchPathol Lab Med 122:447-454, 1998; Barrera J, et al., Arch OtolaryngolHead Neck Surg 126:345-351, 2000) with evidence of improved,recurrence-free survival. Overall, including minor response (25%-50%)tumor shrinkage and reduction of tumor in pathological specimens, over90% responded and the majority had greater than 50% tumor reduction.

[0150] These responses were speculated to be mediated by immuneregression since both B and T lymphocytes were observed infiltrating thetumors. The therapy was not associated with significant toxicity.

[0151] Several unpublished observations serve to document thisspeculation and lead to the present invention.

[0152] 1) Treatment of lymphocytopenic cancer patients with thecombination of NCM has resulted in marked lymphocyte mobilization; whereanalyzed, these patients showed increases in CD45RA positive T-cells(i.e., naive T cels (Table IV).

[0153] 2) Intratumoral or peritumoral injection of NCM in patients withH&NSCC resulted in either reversing immunotherapy-induced tumorregression or in progression of the tumor. The tumor is thus not thesite of immunization.

[0154] 3) Analysis of regional lymph nodes revealed unpublished datawhich indicate that the regional lymph node is the site of immunizationto postulated tumor antigens (see FIGS. 14-18).

[0155] 4) None of these patients treated with NCM developed metastasisexpected in 15% clinically and up to 50% pathologically, indicatingsystemic immunity rather than merely local immunity had been induced.

[0156] 5) Patients were pretested with a skin test to 0.1 ml of NCMprior to treatment. More than 90% of those with a positive skin test(>0.3mm at 24 hours) had robust clinical and pathological response.Patients with negative skin tests had weak or no response. Thus skintesting appears to select good responders.

[0157] Major increases were observed in T lymphocyte counts (CD₂)752→1020 in these T lymphocytopoenic patients (T cell counts 752 vs.normal=1600). Importantly there was a corresponding increase in “naïve”CD45RA positive T cells (532→782). As mentioned previously theseincreases are generally not thought to occur in adults particularly witha pharmacological therapy like NCM. These cells presumably are recentthymic émigrés and could be considered a major new capacity forresponding to new antigens like tumor antigens. The preexisting CD45RApositive cells were not responding to the tumor antigens and may well beincapable of doing so due to the tumor-induced immune suppression(anergy).

[0158] The literature (Hadden J W, Int'l J Immunopharmacol11/12:629-644, 1997; Hadden J W, Int'l J Immunopharmacol 21:79-101,1999) indicates that for both SCC and adenocarcinomas, the two majortypes of cancer, regional lymph nodes reflect abnormalities related tothe tumor, including sinus histocytosis, lymphoid depletion and oftenthe presence of tumor-associated lymphocytes capable of reacting totumor cells (with IL-2). With metastasis lymphoid depletion anddepressed function occur. An unpublished analysis of uninvolved cervicallymph nodes 10 H&NSCC and 10 controls showed reduction in average sizeand an increase in sinus histocytosis associated with H&NSCC (FIGS.14-17).

[0159] Table IV

[0160] Treatment of Lymphocyte Phase Patients with H&NSCC withNCM—Increases in Naïve T Cells in Blood (#/mm³) PATIENT NAÏVE T CELLMARKER PAN T CELL MARKER # PRE POST INCREASE PRE POST INCREASE 1 479 778 +299  704 1171 +467 2 938 1309 +371 1364 1249 −115 3  98  139  +41 146  178  +32 4 341  438  +97  655  590  −65 5 567  652  +97  453  643+190 6 658 1058 +400 1118 1714 +569 7 642 1101 +459  822 1601 +779 MEAN532  782 +250  752 1020 +269

[0161] Following treatment with one cycle of the NCM (IRX-2) protocol(Hadden J W, et al., Arch Otolaryngol Head Neck Surg. 120:395-403, 1994;Meneses A, et al., Arch Pathol Lab Med 122:447-454, 1998; Barrera J, etal., Arch Otolaryngol Head Neck Surg 126:345-351, 2000), the uninvolvedcervical lymph nodes showed the changes indicated in FIGS. 14-17).Compared to the regional lymph nodes of patients with H&NSCC not treatedwith NCM, these nodes showed a significant increase in size, T cell areaand density, and decreases in number of germinal centers and sinushistocytosis and congestion. The lymph nodes of treated patients wereall stimulated and were larger than control nodes with increased T cellarea and density. These nodes were thus not only restored to normal butshowed evidence of T cell predominance, a known positive correlate withsurvival in H&NSCC (Hadden J W. Int'l J Immunopharmacol11/12:629-644,1997).

[0162] Importantly, when the lymph node changes related to B and T cellareas were correlated with the changes in their tumors reflecting T andB cell infiltration, a high degree of correlation was obtained for Tcells (p.<0.01) and B cell s(<0.01) and overall lymphoid presence(p.<0.001). (FIG. 18) In turn, these changes correlate with tumorreduction by pathological and clinical criteria. These findings indicatethat the tumor reactions are directly and positively correlated withlymph node changes and that the tumor reaction reflects the lymph nodechanges as the dependent variable. These findings, taken intoconjunction with knowledge about how the immune system works in general(Roitt I, Brostoff J, Male D. Immunology, J B Lippincott Co, Phila, Pa.,1989), and following tumor transfection with a cytokine gene (Maass G,et al, Proc Natl Acad Sci USA, 1995, 92:5540-5542), indicate that theNCM protocol immunizes these patients to yet unidentified tumor antigensat the level of the lymph nodes. No one has previously presentedevidence for lymph node changes reflecting immunization with autologoustumor antigens. These data convince the applicant that this constitutesa good starting point for trying to induce immunization with previouslyineffective or poorly effective tumor antigens in an effect to yieldregression of distant metastases.

EXAMPLE 3

[0163] Two patients were treated with lymphoma of the head and neck.

[0164] The patients included were those with head and neck cancer whoagreed to participate in the protocol. The following scheme wasfollowed:

[0165] Before treatment, the patients were skin-tested with NCM 0.1 mlsubcutaneously in the forearm, the region was marked, and 24 hrs. laterthe test was read. The test was considered positive if the induction anderythema was equal or larger than 3mm. Each cycle of NCM was for 21 daysas follows: Day 1: Low dose cyclophosphamide (300 mg/m² i.v.) Day 1-21:Indomethacin 25 mg p.o. 3 times daily Zinc sulfate 50 mg p.o. once dailyDay 3-12: NCM 200 units five as 1 ml subcutaneously perilymphatic in theneck.

[0166] Case #1

[0167] The patient was a 23-year-old male who presented on with a priorhistory of three months of the presence of a tumor on the leftsubmaxillary region, with no other symptoms. In the emergency room, hewas found to have lymph adenopathy of the left submaxillary triangle ofapproximately 6.5 cm in diameter of a hard consistency, partially fixedat deep levels. The rest of the physical exam was normal. The incisionalbiopsy showed Hodgkin's lymphoma. The lesion was staged ECIIA. Aone-cycle treatment of NCM was given, obtaining a minor response, as theadenopathy reduced in size by 1 cm in diameter. The biopsy reportobtained after NCM treatment showed 60% of the lesion showed normallymphocytic infiltration, and the rest of the neoplasia (40%) showednecrosis. No viable tumor cells were found.

[0168] Following this, the patient received radiation treatment in theneck of 3600 rads. The patient is currently free of disease.

[0169] Case #2

[0170] The patient is an 82-year-old male, who presented with atwo-month history of a painful mid-neck tumor mass, as well as a 10 kgloss of weight. On physical exam, the patient presented with tumor onthe right palatine tonsil, which was enlarged to approximately 4×3 cm,with an ulcer in the center of the tonsil. On the neck, a rightsubmaxillary lymph node measured approximately 2×2 cm and a lymph nodemass at level 11 and III of approximately 5×5 cm. The rest of the examwas normal. The incisional biopsy of the tonsil and one of the neck'slymph nodes demonstrated defined non-Hodgkin's lymphoma mixed, ofintermediate grade.

[0171] The patient was subjected to two cycles of NCM at the end ofwhich a 1 cm reduction in the diameter of the tonsil and neck adenopathywas observed. The pathological report post-NCM treatment showed livetumor 20%, fragmented and necrotic 30% and normal lymphocyteinfiltration 50%.

[0172] The patient was given chemotherapy (CHOP) for 6 cycles and laterexternal radiotherapy (RT) at a total dose of 4600 rads. He recurred ateight months post RT with adenomegaly at the occipital level. Thepatient died three months later with evidence of neck disease.

EXAMPLE 4

[0173] Ten patients with untreated early stage cervical cancer,clinically staged IB1, IB2 and IIA were treated with local,perilymphatic injections NCM as IRX-2 (10 daily injections) followed byradical hysterectomy at day 21. One day before starting IRX2, patientsreceived a single IV dose of cyclophosphamide at 300 mg/m². oralindomethacin or ibuprofen and zinc sulfate were administered from days 1to 21. The clinical and pathological response, toxicity and disease-freesurvival were evaluated.

[0174] All patients completed NCM treatment and were evaluated forresponse and toxicity. Clinical response was seen in 50% of patients (3partial response (PR), 2 minor response (MR) (>25%<50%reduction)). Sevenpatients underwent surgery, Pathologically tumor reduction associatedwith tumor fragmentation was found in five cases. There was a ratherheterogeneous pattern of cell types infiltrating the tumor whichincluded lymphocytes, plasma cells, neutrophils, macrophages andeosinophils. Treatment was well-tolerated except for mild pain and minorbleeding during injection and gastric intolerance to indomethacin. At a24 months of follow-up, nine patients are disease-free.

[0175] This previously unpublished study shows that peritumoral NCMinduces immune-mediated tumor response in early stage untreated cervicalcarcinoma.

EXAMPLE 5

[0176] Two patients with liver metastasis from primary hepatocellularcarcinoma were treated with intrasplenic NCM (1 or 3 injections). Theprotocol was otherwise as previously described for the H&NSCC, cervical,or lymphoma cases. One patient with advanced hepatocellular carcinomahad a partial response confirmed by tomography, no histology isavailable. The other had a partial response confirmed by surgery.Histological exam showed tumor reduction, fragmentation, and lymphoridinfiltration.

EXAMPLE 6

[0177] Four patients with squamous cell carcinoma of the penis (humanpapiloma virus associated) were treated with the NCM protocol asdescribed above; all four had partial responses clinically and thesurgical specimen showed tumor reduction and fragmentation and lymphoidinfiltration characteristic of the H&NSCC cancer patients.

EXAMPLE 7

[0178] Mice were immunized with PMSA peptides conjugated to ovalbumen100 μg at 3 sites (day 1, 14, and 21) with alum (1:1 Vol) as adjuvant(5@) or NCM (20 units IL2 equivalence) (5@) animals were skin tested atday 28 with ovalbumen (100 pg) (2@) or peptides (100 μg) (3@). Twoanimals treated with ovalbumen plus NCM without peptides responded toovalbumen with positive skin tests. Two animals treated with ovalbumenplus alum did not respond. 2 of 3 animals treated with ovalbumen pluspeptides and NCM responded. None of the animals treated with ovalbumenplus peptides and alum responded. Thus NCM was a superior adjuvant toalum for both tumor peptides and ovalbumen as antigens.

[0179] Throughout this application, various publications, includingUnited States patents, are referenced by author and year and patents bynumber. Full citations for the publications are listed below. Thedisclosures of these publications and patents in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

[0180] The invention has been described in an illustrative manner, andit is to be understood that the terminology which has been used isintended to be in the nature of words of description rather than oflimitation.

[0181] Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the describedinvention, the invention can be practiced otherwise than as specificallydescribed.

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1. A method for unblocking immunization at a regional lymph node by:promoting differentiation and maturation of immature dendritic cells ina regional lymph node and; allowing presentation by resulting maturedendritic cells of antigen to T-cells to gain immunization of theT-cells to the antigen.
 2. A method according to claim 1, wherein saidpromoting step is further defined as administering a natural cytokinemixture (NCM) perilymphatically into lymphatics that drain into lymphnodes regional to a lesion to be treated.
 3. A method according to claim2, wherein the lesion is cancerous or an other persistent lesion.
 4. Amethod according to claim 3, wherein the presented lesion is infectious.5. A method according to claim 1, wherein the antigen is an endogenousantigen.
 6. A method according to claim 1, wherein the antigen is anexogenous antigen.
 7. A method according to claim 2 wherein saidadministering step is further defined as injecting the NCMperilymphatically, intralymphatically, intranodally, intrasplenically,subcutaneously, intramuscularly or intracutaneously.
 8. A method ofinducing immunization to cancer or persistent lesions by administeringan effective amount of an exogenous antigen and an adjuvant consistingof a natural cytokine mixture (NCM).
 9. A method according to claim 7,wherein said administering step is further defined as administering anNCM including IL-1, IL-2, IL-6, IL-8, δIFN and TNFα.
 10. A methodaccording to claim 8 wherein said administering step is further definedas injecting the NCM perilymphatically, intralymphatically,intranodally, intrasplenically, subcutaneously, intramuscularly orintracutaneously.
 11. A method for overcoming mild to moderate T celldepletion and restoring T cell immune response by inducing production ofnaive T cells.
 12. A method according to claim 11, wherein said inducingstep is further defined as administering a natural cytokine mixture(NCM).
 13. A method according to claim 11 wherein said administeringstep is further defined as injecting the NCM perilymphatically,intralymphatically, intranodally, intrasplenically, subcutaneously,intramuscularly or intracutaneously.
 14. A method according to claim 12,wherein said administering step is further defined as injecting an NCMincluding IL-1, IL-2, IL-6, IL-8, δIFN and TNFα.
 15. A method accordingto claim 14, wherein said administering step including administeringabout 150-600 units of IL-2 per injection in the NCM.
 16. A methodaccording to claim 11, wherein said blocking and inducing steps arefurther defined as codelivering cyclophosphamide and a nonsteroidalanti-inflammatory drug (NSAID).
 17. A method of treating a cancer orother persistent lesion in an immune suppressed patient by administeringan effective amount of a natural cytokine mixture as an adjuvant toendogenous or exongenously administering antigen from the cancer orpersistent lesion.
 18. A method according to claim 14, wherein saidadministering step is further defined as injecting an NCM includingIL-1, IL-2, IL-6, IL-8, TNFα and δIFN.
 19. A method according to claim18, wherein said administering step is further defined as injecting anNCM including IL-1, IL-2, IL-6, IL-8, TNFα and 8IFN.
 20. A methodaccording to claim 17, further including the steps of blockingendogenous suppression of T-cells directly or indirectly by theendogenous lesion being treated.
 21. A method according to claim 17,wherein said blocking and inducing steps are further defined ascodelivering cyclophosphamide and a nonsteroidal anti-inflammatory drug(NSAID).
 22. A method according to claim 21, wherein the NSAIDS isselected from the group including indomethacin, ibuprofen, vioxx,celebrex and other related compounds.
 23. A method of vaccineimmunotherapy including the steps of: inducing production of naiveT-cells and exposing the naive T-cells to endogenous or exogenousantigens.
 24. A method according to claim 23, wherein said exposing stepis further defined as exposing the naive T-cells to endogenouslyprocessed peptide preparation resident in regional nodes of a patientwho possesses a lesion.
 25. A method according to claim 24, wherein thelesion is cancerous or infectious.
 26. A method according to claim 23,wherein said exposing step is further defined as administering anexogenously produced antigen.
 27. A method according to claim 23,wherein said antigen is otherwise non-immunogenic peptide.
 28. A methodaccording to claim 23, wherein said exposing step is further defined asimmunizing the naive T-cells with matured peptide presenting dendriticcells at a lymph node distal from a lesion to be treated.
 29. A methodof treating lymphocytopoenic by administering an effective amount of anatural cytokine mixture.